|Year : 2019 | Volume
| Issue : 2 | Page : 93-99
Study on oral microbial flora and antibiotic sensitivity pattern among oral cancer patients in a tertiary cancer care center
Anjali Kanadan1, Arun Anatapadma Bhagwath2, Bastitian Thattil Sebastian3, Parthiban Rudrapathy4, Selvamani Manickam3
1 Department of Microbiology, Mahe Institute of Dental Sciences, Mahe, U.T of Pondicherry, India
2 Yenepoya Research Center, Yenepoya University, Deralakatte, Mangalore, India
3 Department of Oral Pathology & Microbiology, Mahe Institute of Dental Sciences, Mahe, U.T. of Pondicherry, India
4 Department of Microbiology, Malabar Cancer Center, Thalassery, Kerala, India
|Date of Submission||24-May-2019|
|Date of Acceptance||22-Jun-2019|
|Date of Web Publication||29-Jan-2020|
Dr. Arun Anatapadma Bhagwath
Yenepoya Research Center, Yenepoya University, Deralakatte, Mangalore 575018
Source of Support: None, Conflict of Interest: None
Introduction: In recent times, the interaction between cancer and the microbiome has been emphasized. Most studies have focused on the gut microbiota and gastric cancer. However, more attention should be paid on oral microflora as the gastrointestinal tract begins in the oral cavity. The aim of this study is to isolate and identify aerobic microbes present in oral cancer patients and identify their susceptibility to commonly used antibiotics profiles in oral cancer patients. Materials and Methods: This prospective study was carried out in a randomly selected cohort of oral cancer patients at Malabar cancer center Thalasseri for a period of 1 year. Samples were collected from oral cancer patients and subjected for microbiological examination for colony characters, morphology on Gram stain as well for antibiotic sensitivity for different drugs. Results: Of the 96 oral cancer patients, isolated bacterial colonies showed a mixture of gram-positive and gram-negative organisms. Streptococcus species (n = 28) were seen in high number in case of gram-positive organisms, while in gram-negative bacteria (GNB) Klebsiella species (n = 13) was high in number. Among the control group (n = 25), Neisseria flava (n = 11) was the predominant species. All oral cancer patients showed 80% of susceptibility to every class of antibiotics used. Conclusion: This study showed 80% of susceptibility to every class of commonly used antibiotics. But results are not similar in other parts of world. Antimicrobial resistance is emerging among cancer patients. Advancement and monitoring of the microbiota will improve our understanding of the role of the microbiota in carcinogenesis and open new perceptions for future therapeutic and prophylactic modalities.
Keywords: Microbiome, oral cavity, oral cancer, antibiotic
|How to cite this article:|
Kanadan A, Bhagwath AA, Sebastian BT, Rudrapathy P, Manickam S. Study on oral microbial flora and antibiotic sensitivity pattern among oral cancer patients in a tertiary cancer care center. J Orofac Sci 2019;11:93-9
|How to cite this URL:|
Kanadan A, Bhagwath AA, Sebastian BT, Rudrapathy P, Manickam S. Study on oral microbial flora and antibiotic sensitivity pattern among oral cancer patients in a tertiary cancer care center. J Orofac Sci [serial online] 2019 [cited 2020 Jul 13];11:93-9. Available from: http://www.jofs.in/text.asp?2019/11/2/93/276718
| Introduction|| |
Oral cancers arising from lips, tongue, and floor of oral cavity are the subtypes of head and neck cancers. More than 600,000 new oral cancer cases occur every year and the incidence is on the rise causing major global health problems. These cancers are unevenly distributed geographically showing highest incidences in India and France., Poor oral hygiene and use of alcohol and tobacco increase the risk of oral cancers. Oral cavity and oropharynx show abundant anaerobic and aerobic bacteria with predominance of Streptococcus, Bacteroides species (excluding Bacteroides fragilis), Peptostreptococcus species, Prevotella species, Fusobacterium species, Veillonella species, Enterobacteriaceae, and Staphylococci and their successful management depends on the removal of causative agent by selecting proper antibiotics and changing local environment., According to CDC, about one-third of all outpatient antibiotic prescriptions are unnecessary, and it is associated with the development of side effects and drug resistances. Despite the availability of modern diagnostic tests and modern antibiotic treatments, delay in starting early treatment for infections continue to cause more morbidity and mortality.
Serious complications associated with empirical use of antibiotics in immunocompromised patients necessitates proper identification of organisms by culture methods and selection of appropriate antibiotics from antibiotic sensitivity profile for better management and prevention of drug resistance. Emerging resistant organisms may not be detected by a single commercial method in the clinical diagnostic laboratory, hence it is necessary to use conventional broth or agar dilution MIC procedures, special fixed–drug-concentration screening tests, and modified antibiotic interpretive breakpoints to increase the chances of recognizing resistant organisms and their strains. The aim of this study is to isolate and identify aerobic microbes present in oral cancer patients and identify their susceptibility to commonly used antibiotics profiles in oral cancer patients.
| Materials and Methods|| |
This prospective study was carried out in a randomly selected cohort of oral cancer patients at Malabar cancer center Thalasseri from June 2017–June 2018. Ethical clearance was obtained from the Institutional Review Board (Ref. No. 1617/IRB-IEC/13/MCC/06-03-2017/12) before the study. All patients were briefed about the study, counseled, and informed consent was taken.
All consenting patients were diagnosed with oral cancer irrespective of age and gender. Subjects having good oral health, without history or symptoms of oral cancer and pre-cancerous lesions, were taken as control group.
The patients suffering from diabetes mellitus, HIV, hepatitis, and autoimmune diseases were excluded.
Sample collection and processing
Samples were taken by scrubbing the lesions and various regions in the oral cavity using a dry sterile cotton swab and immediately transported to laboratory for microbiological analysis.
The samples were inoculated into brain–heart infusion broth and incubated aerobically at 37°C for 24 hours. After incubation, the broth was inoculated into different media (MacConkey agar, blood agar, chocolate agar) and incubated at 37°C for 24 hours aerobically for bacterial isolation [Figure 1]. The colony characters on the plates and morphology on Gram stain were observed and different isolated colonies were subjected for phenotypic characterization by automated bacterial culture machine BD PhoenixTM (Becton Dickinson).
Antibiotic Sensitivity Test
Antimicrobial susceptibility testing was performed using Kirby-Bauer disk diffusion method on Muller-Hinton agar in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. Antimicrobial susceptibility pattern was determined using eight selected antibiotics from commercial antimicrobial disks (Hi-Media, India) with a wide range of mechanisms of action, including drugs that target cell wall, nucleic acid, and protein [Table 1]. After incubation, the antimicrobial efficacy was determined by measuring the diameter of the zones of inhibition and bacterial strains were classified as susceptible (S), intermediate (I), or resistant (R) depending on the diameter of the inhibition zone.
| Results|| |
Age and gender
Of the 96 cases of oral cancers studied, 70 cases (72.9%) occurred in men and 26 (27.1%) in women, with male-to-female ratio being 2.7:1 [Table 2]. The mean age of occurrence was 56.55 years (range: 32–83 years), and 27 (28.1%) cases were diagnosed in the fifth and sixth decades of life followed by 20 (20.8%) cases in fourth decade of life. Of the 70 male patients, 22 (31.4%) were diagnosed in the fifth decade of their life followed by 20 (28.6%) in their sixth decade, while 6 (8.9%) in the third decade. Of the 26 female patients, 7 (26.9%) occurred in the sixth decade of life followed by 5 (19.2%) with each in the fifth and seventh decades, while 2 (7.7%) occurred in the third and eighth decades [Table 2].
Bacterial isolation from oral cancer patients and controls
Among oral cancer patients, isolated bacterial colonies from oral swabs showed a mixture of gram-positive and gram-negative organisms. Gram-positive organisms isolated were Streptococcus species (n = 28), Coagulase-negative staphylococci (CoNS) (n = 10), Enterococcus species (n = 6), and Bacillus species (n = 12); among these Streptococcus species (n = 28) were predominant in both the genders. In GNB, Klebsiella species (n = 13), Escherichia More Details coli (n = 6), Pseudomonas species (n = 7), and Enterobacter species (n = 5) were isolated [Table 3].
Oral swabs collected for control group (n = 25) showed N. flava (n = 11) as the predominant species followed by Streptococcus mitis, Streptococcus gordonii, Neisseria More Details sicca, and Streptococcus acidominimus (n = 3) [Figure 1].
Streptococcus species distribution
[Figure 2] shows the distribution of Streptococcus species (n = 28) isolated, namely, Streptococcus agalacitiae, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus acidominimus, Streptococcus sanguinis, Streptococcus anginosus, and Streptococcus sorbinus. The predominant Streptococcus species isolated (64%) were S. acidominimus, S. anginosus, and S. sorbinus.
|Figure 2 Distribution of Streptococcus species isolated from oral cancer patients|
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Most commonly found bacteria in various culture media were Staphylococcus species and Klebseilla species [Figure 3].
|Figure 3 Cultures of most commonly found bacteria in oral cancer on Macconkey agar, Blood agar, Chocolate agar. Staphylococcus species (A,B) and Klebsiella species (C).|
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All the bacterial isolates of both gram-positive and gram-negative organisms were subjected to antibiotic sensitivity test by Kirby-Baur disk diffusion method as per CLSI Guidelines.
All gram-negative isolates from oral cancer patients showed 80% susceptibility to every class of antibiotics used. Same susceptibility pattern was also observed in the gram-positive isolates [Table 4] and [Table 5].
|Table 4 Antibiotic susceptibility pattern of Gram positive organism in oral cancer|
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|Table 5 Antibiotic susceptibility pattern of Gram negative organism in oral cancer|
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| Discussion|| |
The oral cavity inhabits mixture of various groups of microbial species with their own dietary and physicochemical requirements. Bacteria are the most predominant microorganisms present, although fungi, viruses, and protozoa are also found. These microorganisms bathe in saliva in oral cavity and saliva may contain approximately 100 million bacteria per milliliter.
Approximately 96% of the oral microbiome belongs to six bacterial phyla Firmicutes, Bacteroides, Proteobacteria, Actinobacteria, Spirochaetes, and Fusobacteria and the remaining 4% belongs to the phyla Euryarchaeota, Chlamydia, Chloroflexi, Synergistetes, Tenericutes, and Candidate. Normally the oral microbiota lives in harmony with the host and maintains homeostatic state, but certain situations can disrupt this relationship and convert to unhealthy state by multifold increase in pathogenic microorganisms.
Oral microbiota may also be associated with the development of cancer; oral cancer patients usually present with poor oral hygiene harboring pathogenic microorganisms, resulting in chronic inflammation of the affected site and the release of large amount of cytokines and growth factors by immune and nonimmune cells may influence carcinogenesis.,,, In general, infection-driven inflammation has been involved in pathogenesis of nearly 13–15% of human cancers, so microorganism causing inflammation should be removed before it causes cancer.,
The microbiome in the oral cavity appears to differ between healthy and cancerous patients, S. anginosus and Treponema denticola are associated with various carcinomas of upper gastrointestinal tract. Shiga et al. suggested that infection by S. anginosus might be implicated in carcinogenesis of squamous cell carcinoma of head and neck in general. Similar results were observed in the present study with a majority of bacterial isolates belonging to S. anginosus and S. sanguinis among oral cancer patients.
Mager et al. investigated the relationship of 40 common salivary bacteria counts between oral cancer patients and healthy controls and observed that Capnocytophaga gingivalis, Prevotella melaninogenica, and S. mitis counts were significantly increased in the oral cancer patients. In our present study, majority of Streptococcus sp. (26% and 38%) were isolated in male and female oral cancer-affected patients, respectively, followed by Klebsiella pneumonia (17%) and Bacillus sp. (18.5%) among male patients while Pseudomonas aeruginosa (19%) and E. coli (11.4%) seen among female patients [Table 2].
Radiation therapy plays an important role in the treatment of head and neck cancer patients, resulting in short- and long-term side effects, including mucositis, osteoradionecrosis, hyposalivation, taste loss, periodontal disease, trismus, and radiation caries. Microbiological studies on the patients undergoing cancer treatment show variation in number and quality of oral microbiota during therapy leading to imbalance in normal oral flora. In the present study, most of the patients had poor oral hygiene due to underlying local factors such as calculus, gingivitis, periodontitis, ulcers, and severe inflammation; hence, we analyzed the patients for antibiotic sensitivity before undergoing radiation therapy.
Gram-positive bacteria and antibiotics
Majority of patients in our study showed gram-positive bacterial growth compared to gram-negative organisms [Table 4]. Most common gram-positive bacteria found among oral cancer patients were Staphylococcus aureus (7), CoNS (11), Streptococcus sp. (28), and Enterococcus sp. (6). These patients were administered with cefoxitin, amikacin, gentamicin, levofloxacin, erythromycin, clindamycin, tetracycline, and co-trimoxazole antibiotics. We found that most microorganisms were resistant to tetracycline, co-trimoxazole, levofloxacin, erythromycin, and cefoxitin, while amikacin showed 100% susceptibility.
Poeschl et al. reported 18% clindamycin and 7% penicillin resistance in aerobes cultured from deep space oral infections. The advantages of clindamycin include oral and parenteral formulations, excellent oral absorption, good distribution into the soft tissue, and its ability to inhibit toxin production and virulence factors in multiple bacterial species. The most common mechanism for development of resistance to clindamycin involves modification of the binding site on the ribosome of the bacteria through methylation that results in concomitant resistance to macrolide antibiotics like erythromycin, clarithromycin, and azithromycin., Thus, a common pattern of clindamycin and erythromycin resistance is often reported as seen in our present study among Streptococcus species [Table 4].
S. aureus was the most common isolate from infections among cancer patients, which may be due to catheter-related bloodstream infections. Methicillin resistance among S. aureus isolates seems to be decreasing in developed countries, but methicillin-resistant S. aureus (MRSA) is still an important cause of morbidity and mortality in cancer patients., In the present study, S. aureus showed 100% susceptibility to amikacin and clindamycin, while low susceptibility to other antibiotics [Table 4].
Gram-negative bacteria and antibiotics
Most frequently isolated three GNB are E. coli, P. aeruginosa, and Klebsiella sp. in neutropenic patients, whereas Citrobacter sp., Enterobacter sp., Proteus sp., and Serratia sp. are less commonly isolated and may vary according to institution.,,, The present study showed maximum number in Klebsiella sp. (12) isolation, while E. coli (6), Pseudomonas sp. (6), Enterobacter sp. (4), and Pasteurella sp. (5) were also isolated. Significant increase in antimicrobial resistance among Enterobacteriaceae has been described recently in cancer.,, In the present study, Enterobacter sp. showed 100% susceptibility to gentamicin, levofloxacin, imipenem, piperacillin, and tazobactum, while low susceptibility to cefepime and amikacin antibiotics. This can be explained by the fact that antibiotic resistance may vary depending on both the type of organism and the type of population studied.
The multidrug resistant pattern among P. aeruginosa is often due to combination of several different mechanisms of drug resistance; multidrug-resistant P. aeruginosa infections in cancer patients have been associated with inadequate therapy, empirical antibiotic therapy, and higher case fatality rates. The increase in carbapenem-resistant GNB, especially Enterobacteriaceae and K. pneumoniae, across the globe is a matter of great concern., Mechanism may be due to either acarbapenem-hydrolyzing enzyme (carbapenemase) or due to changes in outer membrane porins combined with overproduction of AmpC b-lactamases or Extended-spectrum β-lactamases (ESBLs).  The present study showed 100% susceptibility to carbapenems; it can be explained by the fact that these patients are not exposed to carbapenems earlier in life [Table 5].
| Conclusion|| |
Oral cancers occur more commonly in male than in female and predominant bacterial isolates belong to Streptococcus species from oral cancer patients and N. flava from control. All the organisms had 80% sensitivity to the routine antibiotics used.
Although epidemiological reports link between oral carcinogenesis and oral microbial flora, it is postulated that oral bacterial flora does not have a direct play in oral cancer, they can cause comorbidity when they occur in conjunction with other etiological factors such as alcohol and smoking. The clinicians should focus on control of the oral microbes during their treatment strategies rather than elimination.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]